The heterochronic genes of C. elegans regulate the timing of postembryonic developmental events. let-7, which encodes one of the first-described miRNAs, is required for proper differentiation of the adult tissues, and in its absence development is retarded. In mammals, the conserved let-7 miRNA plays important roles in stem cell regeneration and tumorigenesis. In C. elegans, several miRNAs, including miR- 48, miR-241, and miR-84, share identity at the 5'end of the let-7 miRNA, and may target an overlapping set of mRNAs. These let-7 sisters are expressed earlier in development than let-7, and disruption of the three miRNAs together results in early retarded phenotypes. This proposed research explores the regulation and function of mir-48 and is structured around two specific aims. First, a mutation in a cis-regulatory temporal control element of mir-48 will be examined to determine whether it affects mir-48 expression through control of transcription or RNA processing, and provide key new insights into.miRNA biogenesis. The element will be used to identify trans-acting factors involved in this same regulation. A role for lin-28 in miR-48 RNA processing will also be explored, as mammalian Lin-28 was recently shown to inhibit processing of the let-7 miRNA and thereby regulate pluripotency in embryonic stem cells. Second, screens have been conducted to identify new mutations that suppress precocious heterochronic defects resulting from over-expression of mir-48. These screens are expected to identify new heterochronic mutants, miR-48 target genes, and genes involved in miRNA expression and function. 36 independent suppressed lines have been isolated and will be examined through a series of phenotypic analyses and mapped to identify the molecular lesions. The screen has already led to recovery of new alleles of the heterochronic gene lin-66, thereby validating this approach as a powerful means for identification of new regulators of developmental timing. This research will also explore the mechanism of suppression by lin-66. This proposed research will explore how short regulatory RNAs, which are found in the worm C. elegans and in mammals, are controlled during development. These microRNAs regulate myriad biological processes, and the family studied here plays important roles in stem cell identity and cancer progression. In addition, these experiments will identify new genes that regulate the way in which developmental events are programmed to occur in a designated sequence as a complex adult organism forms from a single cell.